Malaria causes an estimated 500 million clinical cases, up to 2.7 million deaths, and a loss of >1% of GDP in Africa annually, and poses a serious concern for travelers and military. Protein Potential's goal is to develop alone or contribute to the development and commercialization of a >90% protective subunit recombinant vaccine for 2 primary markets with a potential for >$1 billion in annual revenues: 1) Travelers from the developed world and 2) Infants and young children in the developing world. The gold standard for such a vaccine is immunization with whole attenuated Plasmodium falciparum (Pf) sporozoites (spz) by the bite of irradiated infected mosquitoes, which protects greater than 90% of experimentally challenged volunteers for at least 10 months. This protection is thought to be primarily mediated by CD8+ T cells, and to a lesser extent CD4+ T cells, against multiple parasite proteins expressed in spz and infected hepatocytes, and secondarily by antibodies against multiple spz proteins. Thus far only immune responses directed against the Pf circumsporozoite protein (PfCSP) have been shown to reproducibly prevent Pf infection in volunteers, but this protection is only about 40% for 2-3 weeks. Thus, there have been intense efforts to identify other spz or liver (pre-erythrocytic) stage proteins that are targets of the highly protective immunity engendered by the irradiated spz vaccine. PfCelTOS, also know as PfAg2, was discovered through analysis of the Pf genome. P. berghei CelTOS has been shown to be involved in spz invasion of the liver, P. falciparum CelTOS was recognized by T cells from 8/8 human volunteers immunized with irradiated P. falciparum spz, and P. yoelii CelTOS protected 64% of mice against infection in the P. yoelii rodent malaria model system when administered as a vaccine. These findings have established PfCelTOS as a prime candidate for being a stand-alone pre-erythrocytic stage Pf vaccine, or part of a multi-protein Pf vaccine designed to elicit protective T cell and antibody responses. Accordingly, we have produced a full length, recombinant PfCelTOS protein in Pichia pastoris, and shown that outbred mice immunized with this protein produce antibodies that recognize Pf spz (titer in indirect fluorescent antibody test, 12,800) and block invasion and development of Pf spz in hepatocytes in vitro (68% inhibition at serum dilution of 1:20). Herein, we propose to optimize production and purification of this protein. Many vaccinologists consider sequential immunization with recombinant virus and recombinant (rec) proteins as the best current approach to optimize antibody and T cell responses in the same recipients. Therefore, we will assess the immunogenicity of the Pf CelTOS rec protein alone with adjuvant, and in a prime boost (sequential immunization) strategy with rec virus expressing PfCelTOS to provide data to support design of Phase II studies in rhesus monkeys and the first clinical trials of these immunogens. In addition, to strengthen the argument for moving the PfCelTOS rec protein to Phase II, we will produce a PyCelTOS rec protein, and conduct studies to achieve significant protective immunity in mice immunized with PyCelTOS immunogens. PUBLIC HEALTH RELEVANCE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Protein Potential's goal is to develop and commercialize a >90% protective malaria vaccine for primary markets with a potential for >$1 billion annual revenues;1) travelers from the developed world, and 2) infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of development and time to market for this malaria vaccine.